Shiga-like toxins (SLTs) are a family of powerful, disease-producing toxins produced by certain strains of Escherichia coli, a type of common bacteria found in humans and animals. SLTs derive their name from the fact that they are cytotoxins similar in both structure and function to Shiga toxin, which is a protein cytotoxin produced by Shigella dysenteriae type 1. This Shigella serotype is responsible for the most severe cases of bacillary dysentery. (SLTs are also known as verotoxins (VTs) because many serotypes that produce this toxin were originally characterized as being vero cell toxinogenic.) The SLT-producing E. coli is a heterogeneous group of bacteria that belong to several different O:H:K serotypes, but they all have in common the ability to discharge one or more SLTs.
SLT-producing E. coli cause a spectrum of diseases in humans from mild, uncomplicated diarrhea and bloody diarrhea to two life-threatening complications, hemorrhagic colitis and hemolytic uremic syndrome (HUS). The life-threatening conditions result from the systemic action of the toxins.
Infants, young children, and the elderly are most susceptible. HUS is a leading cause of acute renal failure in childhood. The syndrome has a fatality rate of about 10%. Up to 30% of HUS survivors develop long-term residual disability, such as chronic renal failure, hypertension, or neurological deficit. In two recent pediatric outbreaks of SLT-producing E. coli in North America, about 7% of symptomatic children developed HUS. In another outbreak in a nursing home in the United States, HUS occurred in 24% of residents, most of whom died. The organism is also showing up in school lunches and, according to the USDA Food Safety and Inspection Service, is a major problem of serious concern. Although information on the incidence of infection is limited, SLT-producing E. coli was recently found to rival Salmonella and Campylobacter as the most common cause of bacterial diarrhea. Karmali, Clin. Microbiol. Rev. 2:15-38 (1989).
Foods of animal origin are the major source of human infection. Although infants, young children, and the elderly are most susceptible, anyone who eats contaminated food can become infected. Infection may also be acquired by person-to-person transmission, which is especially a problem in day care centers and nursing homes.
At least two serologically distinct bacteriophage-mediated SLTs, SLT-I and SLT-II, are involved in human disease. Human isolates of SLT-producing E. coli (SLTEC) elaborate various amounts of either one or both SLT-I and SLT-II. SLT-I is nearly identical, structurally and antigenically, to Shiga toxin. Shiga toxin, SLT-I, and SLT-II are all subunit toxins composed of an enzymatically active A subunit and a number of B subunits that allow binding of the toxin to a specific eucaryotic cell receptor. The toxins kill the cells by inactivating the 60S ribosomal subunit. Binding is followed by internalization of the A subunit, where it inhibits protein synthesis in mammalian cells by inactivating 60S ribosomal subunits through selective structural modification of 28 S ribosomal RNA.
Although SLT-I and SLT-II have a similar subunit structure, bind to the same glycolipid receptor, and inhibit protein synthesis by the same mechanism as Shiga toxin, they also show differences in biological activities in tissue culture and animal models. They also fail to cross-neutralize.
SLT-producing E. coli also causes edema disease (ED) in swine. This disease is a usually fatal condition of weanling pigs, characterized by anorexia, edema of the eyelids, and neurological abnormalities consisting of incoordination and paralysis. E. coli isolates from swine with ED produce an SLT-II related toxin, designated SLT-IIv or VTE. SLT-IIv from edema disease strains differs from SLT-I and SLT-II in that it is much less active on HeLa cells, but very active on Y-1 adrenal cells and Vero cells. The cytotoxicity of SLT-IIv can be neutralized by antiserum to SLT-II. Marques, et al., FEBS Microbiol. Lett. 44:33-38 (1987).
Antibiotics are contraindicated in the treatment of SLT-producing E. coli infection in humans and pigs. Antibiotics actually enhance toxin production by the bacteria. Therefore, their use increases the risk of developing complications such as HUS. To date, treatment of SLTEC infection relies solely on the optimal management of the physiological complications of the infection, i.e., fluid and electrolyte imbalances, anemia, renal failure, and hypertension. Other than that, there is no effective therapy or prevention regimen for the possible development of HUS. Similarly, there is no effective therapy or prevention for swine edema disease.
The only way to control human infection is to address the animal reservoirs. This means instituting practices at the abattoirs and processing plants to reduce cross-contamination between food products from different sources. The USDA and the meat packing industry are thus particularly in need of a rapid test for detecting SLT-producing bacteria.
At present, the toxins are detected by a tedious and time consuming (but highly sensitive) procedure involving the determination of cytotoxicity to cells in culture. The cytotoxicity assay is therefore performed in only a very few centers around the world, mainly reference and research laboratories. Test results take several days to be received.
Moreover, clinical microbiology laboratories and reference laboratories (including the Center for Disease Control and the U.S. Department of Agriculture) rely on the isolation of only a single serotype, 0157:H7, largely because the organism has a phenotypic property (sorbitol negative after 24 h) that facilitates detection from fecal filtrates or mixed flora. However, there are over 50 serotypes of SLT-producing E. coli . Approximately 33 million human fecal samples are tested annually by clinical microbiology labs for pathogens in this country, and an additional 50-100,000 meat samples are tested by the USDA. Therefore, an overall diagnostic strategy, directed toward quickly and effectively detecting the toxin rather than detecting a single serotype, is needed.
A major defense mechanism of humans and animals against pathogenic organisms, such as SLT-producing E. coli, is their ability to produce antibodies that bind to the pathogens and their toxins, inactivating them or preparing them for destruction by specialized cells in the body. Scientists have taken advantage of this fact by inoculating large mammals, such as horses and cows, with various pathogens, toxins, and other antigens, relying on the animal's immune system to produce large quantities of antibodies to the inoculated material. The antibodies are recovered from the animals and then used to treat infections in humans and animals caused by the pathogen or to passively immunize humans and other animals against the pathogen. Such antibodies are also used in immunoassays for the detection of toxins and pathogenic organisms.
Passive immunization also occurs naturally. Humans and many types of animals are born lacking antibodies. They receive some protection by ingesting colostrum and milk from their mother. The colostrum and milk contain appreciable quantities of antibodies and thus are a form of passive immunization.
Cows secrete large amounts of IgG1 immunoglobulin in colostrum and milk. These bovine IgG1 antibodies are relatively protease-resistant and highly homologous with human immunoglobulin G. See, e.g., Lascelles, et al., Transplant. Rev. 19:170-208 (1974) and McLead, et al. Infect. Immun. 44:474-478 (1984).
Several investigators have examined the possibility of using bovine colostrum and milk for passive immunization or treatment of certain diseases in humans and animals. However, the diseases were not caused by SLT-producing E. coli. The inventors are unaware of any attempts to immunize cows with SLTs to produce milk that contains anti-SLT immunoglobulins.
In 1979, Mietens et al. treated 60 infants with acute E. coli gastroenteritis with a bovine milk immunoglobulin concentrate containing antibodies to enteropathogenic strains of E. coli . The concentrate was obtained by hyperimmunization of pregnant cows with the 14 E. coli serotypes that are most frequently responsible for infantile gastroenteritis. Milk obtained during the first 6-8 days of lactation was collected. After skimming, the casein was removed by acid precipitation at pH 4.6 and subsequent centrifugation. The bulk of the lactose and mineral salts was removed by exhaustive ultra-and diafiltration in a reverse osmosis system. The whey protein solution was sterilized by filtration and dried by lyophilization to produce the immunoglobulin concentrate. The results of the study provided evidence that treatment with the milk immunoglobulin concentrate was effective in eliminating enteropathogenic E. coli from the intestine. Mietens, et al., Eur. J. Pediatr. 132:239-252 (1979).
Tacket et al. reported a double-blind, controlled study in which a bovine milk immunoglobulin concentrate with high titers of antibodies against enterotoxigenic E. coli was used as prophylaxis against E. coli challenge in 10 adult volunteers. Lyophilized milk immunoglobulins were prepared from the colostrum of cows immunized with several enterotoxigenic E. coli serotypes, E. coli heat-labile enterotoxin, and cholera toxin. Ten volunteers received the concentrate, and 10 others received a different immunoglobulin concentrate with no anti-E. coli activity. After challenge with enterotoxigenic E. coli, 9 of the 10 controls had diarrhea, but none of the 10 persons who had received the immunoglobulin concentrate against E. coli had diarrhea. Tacket et al., N. Engl. J. Med. 318:1240-1243 (1988).
Several investigators examined the use of bovine milk immunoglobulins for passive immunization against rotavirus infection. Brussow et al. hyperimmunized pregnant cows with four human rotavirus serotypes, resulting in a 100-fold increase in neutralizing milk antibody titers over those of controls. The prepared milk immunoglobulin concentrate consisted of 50% bovine milk immunoglobulins and showed neutralizing activities against the four rotavirus serotypes that were 100 times higher than those in pooled human milk samples and 10 times higher than those in a commercial pooled immunoglobulin preparation from whole human blood serum. The authors proposed that the concentrate be used to induce passive immunity to infantile rotavirus gastroenteritis. Brussow et al., J. Clin. Microbiol. 25:982-986 (1987). Davidson et al. produced a bovine colostrum with a high antibody titer against the four known human rotavirus serotypes and found that it protected susceptible children against rotavirus infection. Davidson et al., The Lancet (Sep. 23, 1989), pgs. 709-712. Archambault et al. inoculated pregnant cows with a bovine rotavirus vaccine and found that the cell-free colostrum isolated from the cows protected newborn calves from a rotavirus challenge exposure on the third day after birth, when they were fed the supplement for the first five days after birth. Archambault et al., Am. J. Vet. Res. 49:1084-1091 (1988).
Boseman-Finkelstein et al. immunized pregnant cows with cholera toxin (CT), a CT-related enterotoxin from E. coli, and Vibrio cholera outer membranes (OMs). Purified colostral immunoglobulin was recovered and examined by various assays. Immunoglobulin preparations were administered orally in infant feeding formula to 6-day old rabbits. Anti-CT and anti-OM immunoglobulins protected against diarrhea in rabbits challenged intraintestionally by virulent cholera vibrios. The protective effects observed were primarily manifested as a delay in the onset of diarrhea disease. Boseman-Finkelstein et al., Infect. Immun. 57:1227-1234 (1989).
U.S. Pat. No. 3,907,987, issued Sep. 23, 1975 to Wilson, discloses the use of a live bacterial vaccine from selected strains of E. coli to vaccinate sows, recover the milk, and feed the milk to newborn pigs to protect against the occurrence of enteric colibacillosis. Essentially the same disclosure is contained in U.S. Pat. No. 3,975,517, issued Aug. 17, 1976 to Wilson.
U.S. Pat. No. 4,816,252 issued Mar. 28, 1989 to Stott et al., discloses a product and process for transferring passive immunity to newborn domestic animals. Immunologically active immunoglobulins are extracted from whey by-product of dairy manufacturing by using ultra-filtration techniques. The patent states that ion exchange techniques can also be used to increase the immunoglobulin concentration in the product. It further states that the dry filtered product can be fed to newborn animals to achieve passive immunity.
The use of antibodies in immunoassays is well known. This includes the use of certain monospecific antibodies. U.S. Pat. No. 4,530,833, issued Jul. 23, 1985 to Wilkins et al. and U.S. Pat. No. 4,533,630 issued Aug. 6, 1985 to Wilkins et al. disclose monospecific antibodies to each of toxin A and toxin B of C. difficile, purification of the antibodies, and their use in assays for C. difficile.
The use of certain receptors for toxins or microorganisms in assays to detect the toxins or microorganisms is also known. For example, U.S. Pat. No. 4,863,852, issued Sep. 5, 1989 to Wilkins et al., discloses the use of a particular carbohydrate structure, which is a receptor for toxin A of C. difficile, to detect toxin A. A specimen is contacted with a reagent containing the receptor, and an assay is conducted to determine if the toxin A is bound to the reagent. The methods for assaying for that binding include the various immunoassays, such as ELISA.
Certain receptors are known for some SLTs. Lingwood et al., J. Biol. Chem., 262:8834-8839 (1987) discloses the binding of verotoxin (SLT) to globotriosyl ceramide (Gb.sub.3) containing the carbohydrate sequence galactose(alpha1-4)galactose(beta1-4)glucose(beta1-1)ceramide. Samuel et al., Infection and Immunity, 58:611-618 (1990) discloses the binding of SLT-I and SLT-II to Gal(alpha1-4)Gal(beta1-4)Glc(beta1-1)Cer (Gb.sub.3). It further discloses that SLT-IIvp (a SLT-II-related variant produced by a porcine isolate) bound to Gb.sub.3, Gal(alpha1-4)Gal(beta1-4)Glc-bovine serum albumin, GalNAc(beta1-3)Gal(alpha1-4)Gal(beta1-4)Glc(beta1-1)Cer (Gb.sub.4), and Gal(beta1-3)GalNAc(beta1-3)Gal(alpha1-4)Gal(beta1-4)Glc(beta1-1)Cer (Gb.sub.5). It also discloses that SLT-IIvh (a SLT-II-related variant produced by a human isolate) bound to Gal(alpha1-4)Gal-bovine serum albumin, Gal(alpha1-4)Gal(beta1-4)Glc-bovine serum albumin, Gal(alpha1-4)Gal(beta1-1)Cer (Gb.sub.2) , and Gb.sub.3. DeGrandis et al., J. Biol. Chem., 264:12520-12525 (1989) discloses that SLT-IIvp binds to Gb.sub.4 and less so to Gb.sub.3. Basta et al., J. Clin. Microbiol., 27:1617-1622 (1989) discloses a sensitive, receptor-specified, enzyme-linked immunosorbent assay (RELISA) to detect SLT-I. GB.sub.3 was de-N-acylated to yield lyso-Gb3, which was more polar but retained SLT-I binding. Lyso-Gb.sub.3 was used to sensitize microdilution plates to bind SLT-I for subsequent immunodetection. The RELISA was used to detect SLT-I in the culture supernatant of a variety of bacteria of known SLT-I status. U.S. patent application Ser. No. 07/211,289 of Lingwood et al., filed Jun. 24, 1988, also discloses SLT receptors and their use in receptor-based binding assays for the detection and quantitation of SLTs.
We have discovered that pregnant cows immunized with purified SLTs produce monospecific, polyclonal antibodies to SLTs that are of a surprisingly and unexpectedly high titer. As a result, we were able to produce very high titer colostrum and milk for use in passive immunization or treatment of SLT toxemia. We also discovered that we were able to immunize the cows with active toxin without ill effect, in contrast to the usual situation in producing polyclonal antibodies where it is necessary to immunize the animal with inactive toxin. This produces antibodies that recognize native epitopes that would not be recognized if inactive toxin were used. As a result of this increased polyvalency, we were able to produce purified IgG that provides outstanding results in terms of increased signal to noise ratio when used as a reagent in assays for the detection of SLTs. The IgG was further purified to provide the monospecific anti-SLT polyclonal antibodies in pure form for use in therapeutic and other special applications. Thus, it is now possible for the first time to rapidly detect the presence of SLTs in samples and to administer anti-SLT antibodies to humans and animals for prophylactic and therapeutic purposes.